Hypertrophic and ischemic heart disease has reached epidemic proportions both in this country and globally. As a result, there is urgent need for new and more effective therapies. Calcineurin is a calcium-regulated protein phosphatase that promotes hypertrophic growth and remodeling of the heart under stress. Our laboratory identified a family of proteins recently renamed RCANs for Regulators of Calcineurin. We demonstrated that RCANs bind to and inhibit calcineurin activity in heart and skeletal muscle. We showed that increased expression of RCAN1 in transgenic mice protects the heart from a wide variety of stresses by blunting both hypertrophic growth and the progression to heart failure after myocardial infarction. Strategies to increase the expression or activity of RCANs in the heart may therefore have clinical value for the treatment and prevention of heart disease. This proposal is a competitive renewal of our RO1 entitled "Modulating Calcineurin Signaling Pathways in Muscle." Accomplishment of our initial goals has contributed significantly toward understanding RCAN's regulation of calcineurin and its biological significance. Our findings have taken us in exciting new directions that may link regulation of calcineurin by RCAN1 to hypoxic or oxidative stress responses. Importantly, we, and others, have recently found that the area of infarct is larger in the hearts of mice lacking RCAN1 after ischemic-reperfusion (I/R). Together these findings suggest that RCAN1 can both limit hypertrophic growth in response to pressure overload and reduce damage from I/R. Our new goals address important questions that have grown out of these findings. Specific Aim 1: To determine the role of the CAATT/enhancer binding protein beta (C/EBPb) in regulating RCAN1.4 expression and test its involvement in hypertrophic remodeling of the heart. In this section we will test the ability of C/EBPb, which is activated in response to cardiac hypoxia, to control RCAN1.4 expression and determine whether C/EBPb activity influences calcineurin signaling or cardiac hypertrophy. Specific Aim 2: To define interactions of RCAN1 isoforms with the ubiquitin/proteasome system and to test whether these contribute isoform-specific functions in the setting of cardiomyocyte hypertrophy. We have identified two different classes of ubiquitin ligase complexes that interact specifically with one RCAN1 isoform but not the other. A cullin 4A E3 ligase complex (Cul4A) interacts with RCAN1.4, whereas the Von Hippel-Lindau factor (VHL), an integral component of hypoxic responses, interacts with RCAN1.1. Specific Aim 3: To define mechanisms through which RCAN1 protects cardiomyocytes from oxidative damage. In this portion of the proposal we will use both in vitro and in vivo techniques to test whether RCAN1 protects cardiac myocytes from oxidative damage and assess the involvement of mechanisms studied in Aims 1 and 2 in this process. These studies are designed to clarify the mechanism, regulation and physiological role of RCAN1 and create the basis for novel therapeutic approaches to the treatment and prevention of heart disease. PUBLIC HEALTH RELEVANCE Cardiovascular disease leading to heart failure is the leading cause of death in the Unites States and is increasing in prevalence despite the application of state-of-the-art therapies. There is a clear need for the development of new therapies. This proposal will provide important information regarding a family of endogenous proteins that protect the heart called Regulators of Calcineurin (RCANs). These studies will clarify the mechanism, regulation and physiological role of RCAN proteins and create the basis for novel therapeutic approaches to regulating calcineurin, a protein involved in the progression of a wide range of pathologies including heart disease and Alzheimers.